Production of rifamycin B

ABSTRACT

Rifamycin B is produced by fermentation by a mutant strain of Streptomyces mediterranei of an aqueous nutrient medium under aerobic conditions.

United States Patent White et a1.

[ Mar. 18, 1975 1 PRODUCTION OF RIFAMYCIN B Inventors: Richard J. White, Como; Giancarlo Lancini, Pavia, both of Italy Assignee: Gruppo [Lepetit S LK, Miianl Italy Filed: Apr. 26, 1973 Appl. No.: 354,811

Foreign Application Prioi'ity Data [56] References Cited UNITED STATES PATENTS 3,150,046 9/1964 Sensi cl All 195/80 R P rYtmtI-y Evaminer-Sa-mih N. Zaharna Assistant Examiner-Robert J. Warden Attorney, Agent, or Firm-Theodore Post; C. Kenneth Bjork Rifamycin B is produced hy fermentation by a mutant ABSTRACT strain of Streptomyces mediterranei of an aqueous nutrient medium under aerobic conditions.

1 Claim, N0 Drawings 1 PRODUCTION OF RIFAMYCIN B BACKGROUND OF THE INVENTION It has previously been reported that during fermentation in normal growth media, Streptomyces mediterranei synthesizes a family of antibiotics collectively referred to as the rifamycin complex (P. Sensi, et al., Antibiotics Annual 1959-1960, page 262). Subsequent work revealed that the addition of sodium diethyl barbiturate to the culture medium resulted essentially in the formation of a single fermentation product, rifamycin B (Margalith P. and Pagani H., Applied Microbiology, 9, 325, 1961).

SUMMARY OF THE INVENTION DESCRIPTION OF SOME PREFERRED EMBODIMENTS The following is a written description of the invention and of the manner and process of making and using it to enable the art skilled to make and use the same and sets forth the best mode contemplated by the inventors of carrying out their invention.

Isolation of Mutant Strain A suspension of spores of Streptomyces mediterranei ATCC 13685 was treated with N-methyl-N'-nitroso-N- nitroguanidine at 1 mg/ml in a pH 9.0 tris(hydroxymethyl)aminomethane buffer for 60 minutes at 28C. The mutagen-treated spores were then washed and plated onto Petri dishes containing Bennett agar. After 14 days incubation at 28C., the surviving colonies were picked off and examined for their ability to produce rifamycin B in liquid medium with and without sodium diethyl barbiturate. The mutant producing high quantities of rifamycin B in the absence of sodium diethyl barbiturate was further examined. Such mutant was called M 18 and deposited with the ATCC under the number 21789 and its characteristics will be described later in detail.

Fermentation An industrial strain of Streptomyces mediterranei ATCC 13685 and strain M 18 were propagated on Bennett agar for 6 to 8 days at 28C. The vegetative medium was inoculated with a slant of Bennett agar (onehalf slant per flask) and the flasks were incubated on a rotary shaker at 28C. for 72 hours. The vegetative medium used contained:

Beef extract 5 g. Yeast extract 5 g. Peptone 5 g. Casein hydrolyzate 3 g. NaCl 1.5 g. H O to make 1 liter medium was used to inoculate (5% v/v) the fermentation medium.

Two fermentation media were used:

RFB 744: a synthetic medium RFB 2244: a complex medium Fermentations were always carried out using m1. of medium in a 500 m1. Erlenmeyer flask.

The composition of the two media was as follows:

RFB 744 Glucose H O pH before sterilizing pH after sterilizing RFB 2244 Glucose Propylene glycol CaCO CuSO .5H O

CoCl .6H O (NHQ Mo O .4H O Sodium diethyl barbiturate pH before sterilization corrected with 15% NaOH pH after sterilization Antifoam A silicone defoamer 0.2 mldper flask containing 50 ml. of growth medium The same media were prepared without the addition of sodium diethyl barbiturate to carry out comparative fermentations. The fermentation conditions and rates I are given in Tables 1 and 2 together with the obtained r The pH was adjusted to 7.3 with NaOH. An amount of ml. vegetative medium was used in 500 ml. Erlenmeyer flasks. After 72 hours growth the vegetative rifamycin B yields.

Table l Rifamycin B in medium, uglml The fermentation medium used was RFB 2244 with and without 1.5 g. of sodium diethyl barbiturate per liter at a temperature of 28C. and a fermentation period of hours. *This result represents an upper limit for the amount of rifamycin B present, as the presence of rifamycin complex in the comparative medium interferes with the normal assay.

Table 2 Rifamycin complex Rifamycin B (#finl) (fig/ml) Strain with without with without barbiturate barbiturate Slre lumyces met itcrranei ATCC 13685 344 107 10 99 Table 2-Continued Rifamycin complex Rifamycin B (pg/ml) (I -g/ml) Strain with without with without barbiturate barbiturate Mutant M 18 ATCC 21789 580 513 10 10 The fermentation medium used was RFB 744 with and without 1.5 g. per liter of sodium diethyl barbiturate. Fermentation time was 144 hours at 28C. on a rotary shaker.

Assay of Rifamycin B The rifamycin B content of the filtered broths was assayed by a differential spectrophotometric technique described by Pasqualucci, et al., Journal of Pharmaceutical Sciences 59, 685, 1970.

Estimation of Rifamycin Complex For the investigation of growth characteristics, Sir. medilerranei and its mutant strain M 18 were grown in a variety of standard media according to Gottlieb and Shirling, (Methods of Characterization of Streptomyl0 ces Species, Intern. J. Syst. Bact. 16, 313-340, 1966);

and, in addition, some media recommended by Waksman were used (The Actinomycetes, Vol. 11, Williams and Wilkins Co., 1961 The following Tables report the growth characteris- 15 tics and properties of the mutant strain M 18 ATCC 21 789 and of Streptomyces mediterranei ATCC 13685.

Table 3.

Assimilation of carbon compounds by .Slreplomyces mednerranez and mutant M 18 Streplomyces Mutant M 18 Carbon Sources medilerranei ATCC 21789 ATCC 13685 25 lnositol -l-l- +1- Fructose 1l- +1- Rhamnose Xylose -ll- +1- Raffinose Arabinose 30 Cellulose Sucrose (positive control) -ll- -+l No carbon (negative control) Table 4 in industrial practice, the product rifamycin B is recovered from a fermented broth in usual ways.

Description of Streptomyces Mediterranei M 18 ATCC 2 l 789 Comparison of morphologic and growth properties of Streptomyces mediterranei ATCC 13685 and mutant M 18 ATCC 2178) Media Streptomyces mediterranei ATCC 13685 Streptomyces medilerranei M 18 ATCC 21789 Yeast extract glucose agar (medium No. 2 No pigmentation of medium. Gottlieb and Shirling) Emerson glucose Abundant growth. yellowish to pink agar orange with rough surface. Scanty aerial mycelium becomes pinkish. No

pinkish. Light amber pigment.

Penassay agar Poor growth.

Pridhams agar Fair growth with smooth surface. Vegetative mycelium h aline to yellowish hitish aerial mycelium with pinkish reverse. Traces Abundant growth yellowish to pink with rough surface. Scanty aerial mycelium.

Good growth. yellowish turning orange yellow. Aerial mycelium becoming Moderate growth; smooth. colorless with lobster red spots. Pink aerial mycelium. No pigmentation of medium.

Starch agar (Medium No. 4 pink. Scarce white aerial mycelium. Gottlieg and Starch hydrolysis: doubtful. Shirling) Dextrose tryptone pigment.

Poor growth, colorless to light orange Abundant growth. orange pink with Abundant growth with smooth surface. Vegetative mycelium greenish to orange with amber shadow. Aerial mycelium absent. Yellow green soluble pigment.

Abundant growth with wrinkled surface. Vegetative mycelium amber brown with orange brown edges. Traces of pinkish aerial mycelium. Deep amber brown soluble pigment.

Abundant growth with smooth surface. Orange vegetative mycelium. No aerial mycelium. Golden yellow soluble pigment.

Abundant growth with very wrinkled surface. Vegetative mycelium amber brown with orange brown edges. Traces of pinkish aerial mycelium. Deep amber brown soluble pigment.

Abundant growth with thin and smooth surface. Vegetative mycelium light orange. No aerial mycelium. Traces of light yellow soluble pigment.

Abundant growth with smooth surface. Vegetative mycelium light orange with small patches of brick red color. Traces of pink aerial mycelium. Yellow soluble pigment.

Abundant growth with smooth surface. Vegetative mycelium deep orange with brownish tinge. Abundant pink aerial mycelium. Chrome yellow soluble pigment. Starch hydrolysis: negative.

Abundant growth with wrinkled surface. Orange vegetative mycelium. No aerial mycelium. Yellow soluble pigment.

Abundant growth with smooth surface. Vegetative mycelium amber rose. Traces of pinkish aerial mycelium. Amber rose soluble pigment.

Table -C ntinued.

Comparison of morphologic and growth properties of Slreplomyces medirerranei ATCC 13685 and mutant M 18 ATCC 21789 Media Streplomyces mediterranei M 18 ATCC 21789 Tyrosine agar (Medium No. 7 Gootlieb and Shirling) Ca malate agar Gelatin Yeast extract molasses agar Czapek-Dox glucose agar Potato agar Glucose asparagine agar Asparagine agar (Medium No. 5 Gottlieb and Shirling) Nutrient agar Nitrate broth Litmus milk Skim milk agar Peptone-yeast extract iron agar (Medium No. (1 (iottlieh and Shirling) Srreptomyces medilerranei ATCC 13685 Poor growth.

Fair growth. colorless. Aerial mycelium whitish with pink tinge. No soluble pigment. Partial digestion of Ca malate.

No pigmentation. Liquefaction: slow and incomplete.

Abundant rough growth colorless to yellowish. whitish aerial mycelium. Deep amber soluble pigment.

Poor growth. thin and colorless to light melon. Traces of pinkish white aerial mycelium. No soluble pigment.

Poor growth. thin and colorless Traces of whitish aerial mycelium.

No soluble pigment.

Fair growth with smooth surface. Thin vegetative mycelium of light orange pink color and yellowish reverse. No aerial mycelium. Some light yellow soluble pigment.

Fair growth with smooth surface. Thin vegetative mycelium of light orange pink color and yellowish reverse. No aerial mycelium. Some light yellow soluble pigment.

Moderate growth with smooth surface; melon to orange with yellowish orange reverse. Aerial mycelium pinkish white. Soluble pigment absent. Surface growth with pinkish aerial mycelium. No reduction to nitrites. Broth becomes yellowish.

No pcptonization or coagulation.

Slight alkaline reaction.

Abundant growth with smooth surface. Vegetative mycelium orange. Casein hydrolysis: positive. Pink aerial mycelium. No soluble pigment. Moderate growth with smooth surface. Colorless vegetative mycelium. Traces of whitish aerial mycelium. No soluble pigment. H- S production: negative. T

pigment.

Abundant growth with smooth surface Amber rose vegetative mycelium.

Abundant growth with smooth surface. Orange vegetative mycelium. No aerial mycelium. Traces of lemon yellow soluble pigment.

Abundant growth with thin and smooth surface. Light orange vegetative mycelium. No aerial mycelium. No soluble pigment.

Abundant growth with smooth surface. Orange vegetative mycelium. No aerial mycelium. Light yellow soluble pigment.

Surface growth with pinkish aerial mycelium. Reduction of nitrates. Broth becomes yellowish.

No coagulation. no peptonization.

Abundant growth with smooth surface. Vegetative mycelium orange. Golden yellow soluble pigment. No aerial mycelium. Casein hydrolysis: strongly positive. Moderate growth with thin and smooth surface. colorlessvggatigg n yc elig No aerial mycelium. No soluble pigment. H 5 production: negative? NOTE: Color determination was performed using the method of Maerz and Paul. Dicti ary ot'Color McGraw-Hill. ln

We claim:

1. In a fermentation process for the production of rifamycin B, the improvement which comprises fermenting under aerobic conditions and in the absence of barbiturate a fermentation medium consisting essentially of an assimilable carbon source, an assimilable nitrogen source and essential mineral salts with a mutant identified as Streptomyces mediterranei M 18 ATCC 21789 until the fermentation medium shows substantial antibiotic activity, and recovering rifamycin B from the fermentation medium. 

1. IN A FERMATION PROCESS FOR THE PRODUCTION OF RIFAMYCIN B, THE IMPROVEMENT WHICH COMPRISES FERMENTING UNDER AEROBIC CONDITIONS AND IN THE ABSENCE OF BARBITURATE A FERMENTATION MEDIUM CONSISTING ESSENTIALLY OF AN ASSIMILABLE CARBON SOURCE, AN ASSIMIAVLBLE NITROGEN SOURCE AND ESSENTIAL MINERAL SALTS WHICH A MUTANT IDENTIFIED AS STREPTOMYCES MEDITERRANEI M 18 ATCC 21789 UNTIL THE FERMENTATION MEDIUM SHOWS SUB STANTIAL ANTIBIOTIC ACTIVITY, AND RECOVERING RIFAMYCIN B FROM THE FERMENTATION MEDIUM. 